536 | Nature | Vol 585 | 24 September 2020
Article
suggest a model (Extended Data Fig. 11d) in which KDM4A multimers
sample pre-existing H3•H3 dimers, and indicates further potential of
precisely mimicking residues such as Bhn to create new speculative
mechanistic models.
Discussion
Our strategy for photoredox-initiation of carbon-centred radical
precursors displays chemoselectivity that is precise enough for
broad installation of previously difficult, even reactive, side chains.
Limitations to our process remain: methyl and aryl radical precur-
sors failed, perhaps owing to higher redox barriers to initiation or
to rapid termination that outstrips addition; however, the pySOOF
system does allow use of HF 2 C•. Notably, we also observed exam-
ples of the site-selective creation of quaternary sites in proteins
through dual carbon-centred radical addition (for example, dual Bn•
to give 4-benzyl,3-phenyl-Aib, BFab, at ~80%; Extended Data Fig. 3a).
Although not the focus of this work, the ability to vary the condi-
tions for the formation of such quaternary centres using longer-lived
carbon-centred radicals with more potent photocatalysts (here
Cat3 > Cat1) to quench/terminate an on-protein α-carbon radical
is an exciting additional observation, which suggests future design
of gem,gem-α,α-bisalkylated motifs known^50 to control stability and
conformation.
Our discovery of new crosslinks using previously inaccessible
unnatural amino acids in proteins (for example, Bhn) suggests that
precise mimicry of residues in PPIs may drive new chemistries,
and hence selectivities. Whereas current crosslinking methods
non-specifically ‘fix’ (for example, formaldehyde, bis-esters), and
hence trap, complexes that are longer-lived or more favoured, our
results here suggest that the future application of crosslinks could
enable the exploitation of reactivity enhanced by effective molarity
(see Supplementary Discussion 6) to precisely cross-react minimal,
size-matched side chains (for example, Bhn) even in transient, reac-
tive interactions. Importantly, in this scenario it is the relative rate
enhancement caused by the environment that is important, rather
than the inherent rate of reactivity. We speculate therefore that better
crosslinking selectivities may now be designed around new amino
acids with unusual chemistries (for example, Williamson ether forma-
tion) and counterintuitively lower (not higher) reactivities that work in
rare, but more information-rich and relevant, contexts (for example,
insertion into precisely matching PPIs that drive effective molar-
ity). Moreover, this crosslinking has enabled complex, activity-based
protein inhibition through the modification of conserved active-site
residues (for example, covalent inhibition of KDM4A by eH3.1-Bhn9).
In this way, one may consider future protein analogues—‘protein cova-
lent inhibitors’—that act akin to emerging, targeted small-molecule
covalent inhibitors^51 , but with enhanced potency and selectivity that
exploits PPIs.
The observed requirement for continual irradiation in our
light-driven approach enables on–off temporal control (Extended
Data Figs. 3e, 7). This suggests not only chemical precision in individual
proteins but also potential future use of defined spatiotemporal irra-
diation^21 ,^52 to control states of protein ensembles over time and site
(for example, tissues). When also paired with in cellulo generation of
Dha^53 , the chemical side-chain versatility that we have observed could
drastically improve our ability to probe complex biological systems
with atomic precision using the tissue-penetrating trigger of light. To
this future end, we have also shown that various biogenic catechola-
mines (Extended Data Fig. 3f ) function in the BACED manifold. This,
along with the endogenous presence of Fe(ii) for pySOOF reactivity,
suggests promise for in vivo reactivity.
Note added in proof: Two relevant papers^ relating to deboronative
radical chain reactions^54 and proximity-based covalent attachment
of small molecules to proteins^55 emerged during the latter stages of
the review of this paper.
Online content
Any methods, additional references, Nature Research reporting sum-
maries, source data, extended data, supplementary information,
acknowledgements, peer review information; details of author con-
tributions and competing interests; and statements of data and code
availability are available at https://doi.org/10.1038/s41586-020-2733-7.- Walsh, C. T., Garneau-Tsodikova, S. & Gatto, G. J., Jr. Protein posttranslational
modifications: the chemistry of proteome diversifications. Angew. Chem. Int. Ed. 44 ,
7342–7372 (2005). - Deribe, Y. L., Pawson, T. & Dikic, I. Post-translational modifications in signal integration.
Nat. Struct. Mol. Biol. 17 , 666 (2010). - Howard, C. J., Yu, R. R., Gardner, M. L., Shimko, J. C. & Ottesen, J. J. Chemical and
biological tools for the preparation of modified histone proteins. Top. Curr. Chem. 363 ,
193–226 (2015). - Yang, A., Cho, K. & Park, H.-S. Chemical biology approaches for studying
posttranslational modifications. RNA Biol. 15 , 427–440 (2018). - Ducry, L. & Stump, B. Antibody−drug conjugates: linking cytotoxic payloads to
monoclonal antibodies. Bioconjug. Chem. 21 , 5–13 (2010). - Hinner, M. J. & Johnsson, K. How to obtain labeled proteins and what to do with them.
Curr. Opin. Biotechnol. 21 , 766–776 (2010). - Leitner, A. et al. Probing native protein structures by chemical cross-linking, mass
spectrometry, and bioinformatics. Mol. Cell. Proteomics 9 , 1634 (2010). - Wang, L., Brock, A., Herberich, B. & Schultz, P. G. Expanding the genetic code of
Escherichia coli. Science 292 , 498 (2001). - Dumas, A., Lercher, L., Spicer, C. D. & Davis, B. G. Designing logical codon reassignment –
expanding the chemistry in biology. Chem. Sci. 6 , 50–69 (2015). - Chin, J. W. Expanding and reprogramming the genetic code. Nature 550 , 53–60 (2017).
- Klemes, Y., Etlinger, J. D. & Goldberg, A. L. Properties of abnormal proteins degraded
rapidly in reticulocytes. Intracellular aggregation of the globin molecules prior to
hydrolysis. J. Biol. Chem. 256 , 8436–8444 (1981). - Chalker, J. M. & Davis, B. G. Chemical mutagenesis: selective post-expression
interconversion of protein amino acid residues. Curr. Opin. Chem. Biol. 14 , 781–789
(2010). - Wright, T. H., Vallée, M. R. J. & Davis, B. G. From chemical mutagenesis to post-expression
mutagenesis: a 50 year Odyssey. Angew. Chem. Int. Ed. 55 , 5896–5903 (2016). - Wright, T. H. et al. Posttranslational mutagenesis: a chemical strategy for exploring
protein side-chain diversity. Science 354 , aag1465 (2016). - Yang, A. et al. A chemical biology route to site-specific authentic protein modifications.
Science 354 , 623–626 (2016). - Tamura, T. & Hamachi, I. Chemistry for covalent modification of endogenous/native
proteins: from test tubes to complex biological systems. J. Am. Chem. Soc. 141 ,
2782–2799 (2019). - Wright, T. H. & Davis, B. G. Post-translational mutagenesis for installation of natural and
unnatural amino acid side chains into recombinant proteins. Nat. Protoc. 12 , 2243–2250
(2017). - Sletten, E. M. & Bertozzi, C. R. Bioorthogonal chemistry: fishing for selectivity in a sea of
functionality. Angew. Chem. Int. Ed. 48 , 6974–6998 (2009). - Imiołek, M. et al. Selective radical trifluoromethylation of native residues in proteins.
J. Am. Chem. Soc. 140 , 1568–1571 (2018). - Isenegger, P. G. & Davis, B. G. Concepts of catalysis in site-selective protein modifications.
J. Am. Chem. Soc. 141 , 8005–8013 (2019). - Lim, R. K. V. & Lin, Q. Photoinducible bioorthogonal chemistry: a spatiotemporally
controllable tool to visualize and perturb proteins in live cells. Acc. Chem. Res. 44 ,
828–839 (2011). - Twilton, J. et al. The merger of transition metal and photocatalysis. Nat. Rev. Chem. 1 ,
0052 (2017). - Prier, C. K., Rankic, D. A. & MacMillan, D. W. C. Visible light photoredox catalysis
with transition metal complexes: applications in organic synthesis. Chem. Rev. 113 ,
5322–5363 (2013). - Bloom, S. et al. Decarboxylative alkylation for site-selective bioconjugation of native
proteins via oxidation potentials. Nat. Chem. 10 , 205 (2018). - Yu, Y. et al. Chemoselective peptide modification via photocatalytic tryptophan
β-position conjugation. J. Am. Chem. Soc. 140 , 6797–6800 (2018). - de Bruijn, A. D. & Roelfes, G. Chemical modification of dehydrated amino acids in natural
antimicrobial peptides by photoredox catalysis. Chem. Eur. J. 24 , 11314–11318 (2018). - Povie, G. et al. Catechols as sources of hydrogen atoms in radical deiodination and
related reactions. Angew. Chem. Int. Ed. 55 , 11221–11225 (2016). - Matsui, J. K., Lang, S. B., Heitz, D. R. & Molander, G. A. Photoredox-mediated routes to
radicals: the value of catalytic radical generation in synthetic methods development.
ACS Catal. 7 , 2563–2575 (2017). - Robole, Z. M., Rahn, K. L., Lampkin, B. J., Anand, R. K. & VanVeller, B. Tuning the
electrochemical redox potentials of catechol with boronic acid derivatives. J. Org. Chem.
84 , 2346–2350 (2019). - Ghosh, T. et al. Mixed-ligand complexes of ruthenium(ii) containing new photoactive or
electroactive ligands: synthesis, spectral characterization and DNA interactions. J. Biol.
Inorg. Chem. 10 , 496 (2005). - Dolbier, W. R. Structure, reactivity, and chemistry of fluoroalkyl radicals. Chem. Rev. 96 ,
1557–1584 (1996). - Zhang, L., Dolbier, W. R., Sheeller, B. & Ingold, K. U. Absolute rate constants of alkene
addition reactions of a fluorinated radical in water. J. Am. Chem. Soc. 124 , 6362–6366
(2002).